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Monocyte chemoattractant protein 1 and interleukin 8 production by rheumatoid synoviocytes. Effects of anti-rheumatic drugs
MONOCYTE CHEMOATTRACTANT PROTEIN 1 AND INTERLEUKIN 8 PRODUCTION BY RHEUMATOID SYNOVIOCYTES. EFFECTS OF ANTI-RHEUMATIC DRUGS Pius Loetscher,’
Beatrice Dewald,2 Marco Baggiolini,2
Michael Seitz’
Activated synoviocytes are major effector cells in the pathogenesis of rheumatoid arthritis (RA) because of their capacity to secrete a variety of inflammatory mediators. Among these mediators, the chemotactic proteins monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-S) are likely to contribute to the recruitment of inflammatory cells into the arthritic joint. We examined the effects of anti-rheumatic drugs on the MCP-1 and IL-8 production by cultured RA synoviocytes exposed to pro-inflammatory agonists. Both chemotactic cytokines were quantified by specific enzyme-linked immunosorbent assays (ELISA), and found to accumulate in the culture supernatants. Although the time course of formation was similar, the yield of IL-8 was three to IO-fold higher than that of MCP-1. Non-steroidal anti-inflammatory drugs inhibited the synthesis of prostaglandins, but did not influence the production and release of both chemotactic cytokines. Of three disease-modifying drugs tested, dexamethasone and gold sodium thiomalate (GST) inhibited the production of IL-8 and MCP-1, while methotrexate (MTX) was inactive. Dexamethasone reduced the production of MCP-1 and IL-8 by 2045% and 60-SO%, respectively, whilst GST inhibited MCP-1 and IL-8 synthesis in suboptimally, but not in optimally stimulated synoviocytes. Taken together, these results show that the production of MCP-1 and IL-8 is similarly affected by anti-rheumatic drugs and that dexamethasone is the most potent inhibitor suggesting that part of the anti-rheumatic action of glucocorticoids is due to prevention of accumulation of chemotactic cytokines acting on neutrophils and monoctyes.
Rheumatoid arthritis (RA) is a chronic articular inflammatory disorder characterized by synovial lining cell hyperplasia and infiltration of inflammatory cells.’ Synoviocytes of the hyperplastic lining layer exhibit an activated phenotype, and are believed to contribute to the invasive and erosive properties of the inflamed tissue. Cultured synoviocytes exposed to cytokines, such as interleukin 1 (IL-l) and/or tumour necrosis factor (Y (TNF-a), secrete a variety of mediators of inflammation and tissue destruction
From the ‘Department of Rheumatology, University of Bern, Inselspital, CH-3010 Bern, Switzerland, and ‘Theodor-Kocher Institute, University of Bern, CH-3000 Bern, Switzerland. Correspondence to: Dr Pius Loetscher, Department of Rheumatology, University of Bern, Inselspital, CH-3010 Bern, Switzerland. Received 21 April 1993; revised and accepted for publication 12 August 1993 0 1994 Academic Press Limited 1043-4666/94/020162+09 $0800/O KEY WORDS: Anti-rheumatic thritiskynoviocytes 162
drug/IL-S/MCP-l/Rheumatoid
ar-
including collagenase,2-4 plasminogen activator,5 prostaglandin E2 (PGE2),2,4’5 interleukin 6 (IL-6),6 granulocyte-macrophage colony-stimulating factor (GM-CSF)“’ and granulocyte-CSF (G-CSF) ,7 and show increased proliferation.4,9 In addition, several recent studies have demonstrated that synoviocytes also release interleukin 8 (IL-8)10-13 and monocyte chemoattractant protein-l (MCP-1)14’15 in response to cytokine stimulation. IL-8 and MCP-1 belong to a family of structurally related, inducible proteins with four conserved cysteine residues. l&l7 The family is divided into two subfamilies according to the position of the first two cysteines which can be adjacent as for MCP-1 (CC) or separated by one amino acid as for IL-S (CXC). MCP1 and IL-S have different target cell selectivity. IL-8 acts preferentially on neutrophils,i’ whereas MCP-1 activates monocytes and basophils. 19-21 These biological properties together with the finding that MCP1 and IL-S are present at elevated concentrations in RA joint fluids15,22,23 suggest that these chemoattractants contribute to the recruitment of monoCYTOKINE,
Vol. 6, No. 2 (March), 1994: pp 162-170
Effects of anti-rheumatic
nuclear and polymorphonuclear phagocytes into the inflamed synovium. Modulation of MCP-1 and IL-8 production is, therefore, likely to influence disease activity. Non-steriodal anti-inflammatory drugs (NSAID), glucocorticoids, methotrexate (MTX) and gold compounds are widely used to treat rheumatoid arthritis. The mechanisms of action of these drugs is only partly understood. While NSAID act primarily by inhibiting anti-inflammatory cyclo-oxygenase,24 additional effects may be attributed to interference with proinflammatory cytokine production.25-29 Glucocorticoids inhibit production of prostanoids by blocking phospholipase A2,30 but also regulate the expression of a variety of genes.31’32 The mechanism of the disease modifying effects of MTX and gold compounds is still unclear .33-35 We have recently demonstrated that the production of IL-8 by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis is reduced by dexamethasone, MTX and gold sodium thiomalate (GST), and slightly increased by indomethacin.36,37 We have now studied the effects of a number of representative anti-rheumatic drugs on the production of IL-8 and MCP-1 by stimulated synoviocytes. Furthermore, we examined whether these two closely related chemotactic proteins are differentially modulated by the action of these agents.
I
I
Figure
1.
I
I
I
1
0
Standard
of Antigenic
Production of MCP-1 and IL-8 by RA Synoviocytes In agreement with former reports,lO~ll~ l5 we found that synoviocytes in culture release MCP-1 and IL-8 upon stimulation with LPS, IL-l&, IL-ll3 and
I,
1
curve
of the sandwich
I
4 ELBA
I
5 for MCP-1.
Each point represents the mean of 22 separate determinations carried out in duplicate and vertical bars indicate standard deviation. Since colour development was allowed to proceed for various periods of time, the optical density values at 405 nm were normalized to the absorbance obtained with 5 rig/ml MCP-1. 100% correspond to values ranging from 1.79 to 2.48. The lower limit of detection was 37 p&/ml (P = 0.02). Identical standard curves were obtained with chemically synthesized and recombinant MCP-1.
1.
Specificity 0
MCP-1
A standard curve representing a compilation of 22 separate assays is shown in Fig. 1. The lower limit of detection of MCP-1 was about 40 pg/ml. To assess selectivity, several structurally related cytokines as well as IL-1B and TNF-or were tested for crossreactivity. As shown in Table 1, none of the control cytokines was detected by the sandwich ELISA. Similar negative results were obtained in a direct ELISA (data not shown). The reproducibility of the assay is indicated by the coefficients of variation of absorbance at different MCP-1 concentrations, which ranged from 4.6 to 6.6% and 9.7 to 16.4% in intra- and interassay analyses, respectively.
I
2 3 MCP-1 (rig/ml)
of sandwich
Cytokines
Detection
/ 163
100
TABLE
RESULTS
drugs on MCP-l/IL-8
0.1
Absorbance 1.0
ELISA (405 nm)d 5.0
for MCP-1. 10.0
50.0
0.142 MCP-1 RANTES MIP-la MIP-ll3 IL-8 PF4 CTAP-III NAP-2 GROW GROP GROr IPlO IL-1p TNF-a a The cytokine
0.169 0.156 0.141
0.385 0.140 0.142
0.152 0.155 0.144 0.150 0.148 0.148 0.146 0.149
0.155 0.149 0.148 0.150 0.150 0.147 0.146 0.150
0.139 0.145 0.140 0.147 0.141 0.146 0.154 0.141 0.142 0.142 0.146
0.148
0.144
2.089 0.148
0.137 concentration
(~/ml)
is indicated.
TNF-a. Both chemotactic cytokines steadily accumulated in the culture supernatants for up to 10 days as shown in Fig. 2 for synovial cells stimulated with ILll3 and TNF-a. Similar results were also obtained with IL-la and LPS (data not shown). Maximal levels of MCP-1 and IL-8 were released after stimulation with
164 I Loetscher
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et al.
T
Vol. 6, No. 2 (March 1994: 162-170)
tially modulate the production of colony-stimulating factors in synovial fibroblasts.42 Therefore, we examined whether common NSAID with different plasma half-lives (tiaprofenic acid < indomethacin < naproxen < piroxicam43) also affect the release of IL8 and MCP-1. As shown in Fig. 3, all NSAIfi strongly decreased PGE production without appreciably affecting the release of MCP-1 and IL-K Although the yield of both cytokines tended to rise with the concentration of NSAID added, the increase was not statistically significant. MCP-1 and IL-S release by stimulated synovial cells were also unaffected by the addition of exogenous PGE,, even at high concentrations (data not shown). These results indicate that cycle-oxygenase products are not involved in the regulation of these two chemotactic cytokines in synoviocytes, and that NSAID do not modulate MCP-1 and IL-8 production.
Effect of Dexamethasone
0 Figure
2.
Time
2 course
4 of MCP-1
6 Days and IL-8
8 release
by
10 synoviocytes.
RA synoviocytes were cultured in RPMI-1% FCS in the absence (A) or presence of 10 rig/ml IL-lp (0) or TNF-ol (m). MCP-1 and IL-S were determined in the culture supernatants by ELBA. Mean values k SEM for cells from three RA patients.
l-10 rig/ml IL-1 or TNF-(r. These concentrations are closely comparable to those observed in synovial fluids from patients with RA.38-40 IL-l was always more potent than TNF-a (Fig. 2) or LPS. Synoviocytes produced about three to 10 times more IL-S and MCP-1 irrespective of the inducing agent. As formerly reported for IL-8,41 MCP-1 was found to be remarkably resistant to proteolytic degradation. Over 95% of antigenic MCP-1 added to synovial cell cultures could be recovered after 10 days of incubation (data not shown).
Effect of Non-steroidal
Anti-inflammatory
Drugs
NSAID are still the mainstay of therapy for rheumatoid arthritis. It has been shown that they differen-
Glucocorticoids are potent anti-inflammatory agents and are often used intra-articularly to treat synovitis. Hence, the effect of the synthetic glucocorticoid dexamethasone on the expression of IL-S and MCP-1 by synoviocytes in response to suboptimal (0.01 rig/ml) and optimal stimulatory concentrations (1 rig/ml) of IL-la and -p, TNF-a and LPS was investigated. As shown in Fig. 4, addition of dexamethasone (0.01 and 1 PM) significantly decreased the production of both cytokines. The inhibitory effect was similar for all stimuli and concentrations used. The production of IL-8, however, was inhibited more markedly than that of MCP-1 (P < 0.05; Fig. 4). These results together with the data obtained with NSAID suggest that the inhibitory action of dexamethasone is not mediated by a change in prostanoid release, but rather by effects on gene expression.
Effect of Methotrexate Thiomalate
and Gold Sodium
Treatment of synoviocytes with increasing concentrations of methotrexate followed by stimulation with suboptimal (0.01 rig/ml) or optimal concentrations (1 rig/ml) of IL-lp did not affect the production of either MCP-1 or IL-S (Fig. SA). MTX concentrations corresponding to serum levels observed therapeutically (0.01 and 1 pg/ml)44 as well as an excessive concentration (50 kg/ml) were tested. In contrast, GST (l-50 pg/ml) significantly reduced the production of both MCP-1 and IL-8 in RA synoviocytes stimulated with a suboptimal concentration of IL-lp (Fig. 5B). Inhibition was observed at concen-
Effects
A
of anti-rheumatic
drugs
on MCP-l/IL-8
I 165
100, Stimulus:
0.01
ngiml
II
Stimulus:
1.0 ngiml
Stimulus:
0.01 ngiml
Stimulus:
1.0 rig/ml
150 g $ z8 c
100
2 B
50
I””
0
hi z 100 2
e Figure 3. Effect of NSAID on MCP-1, l@ stimulated RA synoviocytes.
0 IL-8
and PGE release
by IL-
The cells were cultured for 1 h in RPMI-10% FCS with or without drug before addition of IL-lp (10 rig/ml). After 72 h MCP-1, IL-8 and PGE were assayed in the culture supernatants. Percent release with respect to control (no NSAID) is shown: 0,O.l PM; q , 1 PM; q , 10 PM; 0, 100 pM. Control values (100%) were 9 to 47 rig/ml for MCP-1, 73 to 217 &ml for IL-S and 6 to 26 @ml for PGE. Mean values + SEM for cells from four RA patients. t P < 0.05 or 0.02, * P < 0.005 or 0.001.
None IL-la Figure 4. Effect of dexamethasone 8 release by RA synoviocytes.
IL-1S (dex)
TNF-(Y LPS on (A) MCP-1 and (B) IL-
The cells were cultured for 2 h in RPMI-1% FCS with or without dexamethasone (0.01 or 1 KM) before addition of indicated stimulus (0.01 or 1 q/ml). After 72 h MCP-1 and IL-S concentrations were determined in the culture supernatants. Percent release with respect to control (no dex) is shown: q , 0.01 PM; 8,l.O ~.LM. Mean values k SEM for cells from four RA patients. P < 0.05 for MCP-1 release and P < 0.005 for IL-8 release.
166 I Loetscher
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et al.
MCP-1
0.01
1
0.01
1
IL-1B (ngiml) Figure 5. late (GST) synoviocytes.
Effect of methotrexate on MCP-1 and IL-8
(MTX) release
and gold sodium thiomaby IL-@ stimulated RA
The cells were cultured for 2 h in RPMI-1% FCS with or without MTX or GST and then stimulated with 0.01 rig/ml or 1 rig/ml IL-1B. 0.01 pg/ml; ma.1 pglml; g, SO kg/ml MTX. (B) q . 10 kg/ml; B. 50 kg/ml GST. After 72 h MCP-1 and IL-8 were assayed in the culture supernatants. Percent release with respect to control (no drug) is shown. Control values (100%) were as follows: 0.01 nglml IL-l@, 9-18 ngiml MCP-1 and 26-143 ngiml IL-S; 1 ng/ ml IL-ll3,20-45 rig/ml MCP-1 and 194-273 rig/ml IL-8. Mean values + SEM for cells from four RA patients. Significant differences are indicated (t. P < 0.02; $, P < 0.01; #, P < 0.001).
trations that are attained in the serum and synovial tissue of GST-treated RA patients.45%46 The effect on IL-8 production was more pronounced than that on MCP-1. No inhibitory effect, however, was detected when the cells were exposed to a IL-lp concentration that resulted in maximal release of the cytokines (Fig. 5B).
DISCUSSION This study was designed to explore the potential role of commonly used anti-rheumatic drugs in reduc-
Vol. 6, No. 2 (March 1994: 162-170)
ing the production of MCP-1 and IL-S by RA synovial cells. MCP-1 and IL-8 are major mediators of inflammation and joint destruction released, by activated synoviocytes in the proliferating synovium. Their main biological function, leukocyte chemotaxis and activation,16’18’47 suggest that they play an important role in the pathogenesis of rheumatoid arthritis. Having confirmed that synoviocytes produce substantial amounts of MCP-1 and IL-8 in response to pro-inflammatory agonists, we studied the effects of anti-rheumatic drugs. NSAID inhibited prostaglandin synthesis in a concentration dependent fashion, but they had no significant effect on MCP-1 and IL-S production. Similarly, it has been reported that NSAID did not affect MCP-1 mRNA levels in mouse fibroblasts.48 In peripheral blood mononuclear cells, however, IL-8 production was increased in the presence of indomethacin.36 Recent studies have reported that various NSAID inhibit the production of other proinflammatory cytokines including IL-l, TNF-a and IL-6 by activated mononuclear phagocytes,25-29 and differentially regulate the levels of colony-stimulating factors in synovial fibroblasts.42 Together, these findings suggest that the actions of NSAID are cell-type and cytokine specific. Some of the effects of these drugs may be mediated by a decrease of prostanoid levels, while others appear to be unrelated to inhibition of cycle-oxygenase. In fact, in the present study added PGE2 did not influence MCP-1 and IL-8 production by synoviocytes and only minimal effects on IL-8 expression by lipopolysaccharide-stimulated alveolar macrophages were observed by others.49 On the other hand, PGE2 inhibited the expression of IL-8 at the mRNA and protein level in peripheral blood monocytes.49 In contrast to NSAID, the synthetic glucocorticoid dexamethasone suppressed the production of MCP-1 and IL-8 by RA synoviocytes exposed to proinflammatory cytokines or bacterial endotoxin. The ineffectiveness of NSAID suggests that the inhibition by dexamethasone is mediated at the level of gene expression rather than indirectly through prostanoids. Glucocorticoids have recently been shown to inhibit the induction of MCP-1 by IL-l or TNF-u in human fibrosarcoma cells5’ and by serum and dibutyryl CAMP in human synoviocytes.‘4 Similarly, glucocorticoids blocked the induction of JE, the murine homolog of MCP-1, by serum or platelet-derived growth factor in cultured vascular smooth muscle cells’i and 3T3 fibroblasts.48 Several mechanisms for the regulation of MCP-1 gene expression by glucocorticoid have been suggested including mRNA destabilizatiot?l and/or inhibition of transcription.48,50 Glucocorticoids also inhibit the expression of IL-8,52,53 an effect that was attributed to inhibition of gene transcription in a human fibrosarcoma cell line.53
Effects of anti-rheumaticdrugs on MCP-l/IL-S / 167
MTX is a highly effective drug for the treatment of RA.54 Yet, we did not observe any significant regulatory activity of this drug on the production of MCP1 and IL-8 by RA synoviocytes. Likewise, no effect on IL-8 release from endothelial cells has been reported.52 In PBMC from RA patients, we previously observed an inhibition of IL-8 production by MTX3’ in contrast
to Deleuran
et al. who found
no sup-
pression of IL-8 secretion.52 It was recently reported that MTX inhibits the activity of IL-1 released from stimulated human PBMC without affecting IL-1 production.29,55 Several effects may contribute to the action of MTX, but the mechanism of its anti-
rheumatic activity remains a mystery.56 Several effects of gold compounds, such as GST, on monocytes have been reported. They include inhibition of accessory cell function,57 differentiation into effector cells,58 decreased production of complement
components59 and decreased expression of HLA-DR in response to IFN. 6o In addition, GST was shown to inhibit IL-l activity29 and IL-8 release.37 Monocytes, however, are not the only target of GST. As shown by the present work, GST, at concentrations that are attained in the serum and synovium of RA patients,
significantly decreased MCP-1 and IL-8 release from synoviocytes under moderate stimulatory conditions (0.01 rig/ml IL-lp). GST has previously been shown to inhibit the proliferation of endothelial cells61 fibroblasts and lymphocytes,62,63
and to modulate
IL-8
mRNA expression and protein secretion.52 These studies indicate that GST affects a variety of cell types and different cellular activities. The biochemical basis for these various effects, however, has still to be elucidated.34 In conclusion, among several anti-rheumatic drugs examined, dexamethasone exhibited the most potent inhibitory effect on the production of MCP-1 and IL-8 by synoviocytes derived from patients with rheumatoid arthritis. A decrease in the synthesis of chemotactic proteins in the inflamed synovium may be one of the mechanisms by which glucocorticoids exert their anti-inflammatory
rheumatoid
MATERIALS
actions in diseases such as
arthritis.
AND METHODS
Reagents Penicillin-streptomycin-fungizone, RPMI-1640, Escoli lipopolisaccharide (LPS), and trypsin-EDTA were purchased from Gibco BRL (Gaithersburg, MD), protein G Sepharose was from Pharmacia-LKB (Uppsala, Sweden), alkaline phosphatase-conjugated goat anti-rabbit antibodies and alkaline phosphatase substrate kit were from Bio-Rad Laboratories AG (Richmond, CA). Fetal calf
cherichia
serum (FCS) was obtained from Amimed (Muttenz, Switzerland). Dexamethasone, prostaglandin EIz,indomethacin, and collagenase type II from Clostridium histolyticum were obtained from Sigma (St Louis, MO). Naproxen was supplied by Syntex Laboratories, piroxicam by Pfizer (Groton, CT), gold sodium thiomalate by Byk Gulden (Byk AG, Kreuzlingen, Switzerland) and methotrexate by Cyanamid AG (Zurich, Switzerland). Streptavidin-alkaline phosphatase, NHS-LC-biotin was purchased from Pierce (Rockford, IL) and MaxiSorb microtitre plates were from Nunc (Roskilde, Denmark). IL-& NAP-2, CTAP-III, PBP, Grocu, B, y, PF4, IPlO and MCP-1 were chemically synthesized and generously provided by Dr I. Clark-Lewis, University of British Columbia, Vancouver, Canada. Recombinant MIPlo, MIP-ll3 and RANTES were kindly provided by Dr C.A. Dahinden, Department of Clinical Immunology, University of Bern, Bern, Switzerland, with the permission of T. Schall, Genentech Inc., So. San Francisco, USA. IL-la and IL-ll3 were generously gifts from Dr K. Vosbeck, CibaGeigy AG, Basel, Switzerland, and TNF-or was kindly supplied by Hoffmann-La Roche, Basel, Switzerland.
Synovial Cell Cultures Synovial tissue was obtained from RA patients undergoing total shoulder, knee or hip joint replacement, who were selected according to the American Rheumatoid Association criteria. 64 Fresh synovial tissue was minced and digested in RPM1 1640 medium containing 1% fetal calf serum (FCS), penicillin-streptomycin-fungizone and 1 mg/ ml collagenase at 37°C until dissolution of the tissue fragments. After filtration through a nylon mesh, the cells were washed with phosphate-buffered saline (PBS) and cultured in RPM1 1640 supplemented with 10% FCS and antibiotics in a humidified COz atmosphere. The following day, nonadherent cells were removed and the medium was replaced. Upon reaching confluence, the cells were harvested with trypsin/EDTA, and subcultures were established. The content of macrophage-like cells determined by staining for non-specific esterase65ranged from 3 to 13%. Synoviocytes from passage 3 to 6 were used for all experiments. They were transferred to 24-well plates (lo5 cells per well) in RPM1 1640 containing 10% FCS. After 24 h, the cells were washed twice with PBS and incubated with 1 ml RPM1 1640 supplemented with 1% FCS (if not otherwise indicated). Drugs were added to the cultures 1 or 2 h before stimulation with pro-inflammatory agonists. NSAID, PGEz and dexamethasone were dissolved in ethanol. The final ethanol concentration did not exceed 0.1% which had no effect on synoviocyte viability as assessedby trypan blue exclusion. Culture supernatants collected at the indicated times were stored -70°C until assayedfor IL-8, MCP-1 and PGE.
Anti-MCP-1
Antibodies
Rabbit polyclonal antibodies were obtained by intradermal injection of synthetic MCP-1 (220 kg) emulsified with complete Freund’s adjuvant followed by three booster injections at weekly intervals. Anti-MCP-1 antibodies in the serum were determined by a direct ELISA. Microtitre plate
168 I Loetscher
CYTOKINE,
et al.
wells (Nunc-Immuno Plate Maxisorb) were coated with 100 ng MCP-1 in 100 ~1 PBS, pH 7.2, overnight at 4°C. After blocking non-specific binding sites with 300 ~1 1 mg/ml BSA in PBS for 30 min at 37”C, the plates were rinsed three times with wash buffer (TBS-Tween: 0.05 M tris-HCl, 0.15 M NaCl, pH 7.5, containing 0.05% Tween 20 and 0.02% sodium azide), and incubated with 100 pi/well of antiserum serially diluted in TBS-Tween. Following incubation at 37°C for 90 min, the plate was washed twice with wash buffer and 100 ~1 of a 1:2000 dilution of alkaline phosphatase conjugated to goat anti-rabbit IgG in wash buffer were added. The plate was incubated for 30 min at 37°C and, after five washes with TBS (without Tween 20), 100 ~1 of substrate solution (1 mg/ml p-nitrophenylphosphate in diethanolamine buffer) was placed in each well. After optimal colour development at 37”C, the reaction was stopped by the addition of 100 ~1 0.4 M NaOH and the absorbance was measured at 40.5 nm in an ELISA reader. The IgG fraction of high titre antiserum (1:lOOOOO) was isolated by chromatography on protein G Sepharose. For use as detecting antibody, a portion of the purified IgG fraction was biotinylated with NHS-LC-Biotin according to the manufacturer’s instruction. Unreacted NHS-LC-biotin was removed by centrifugation using a Centricon 30 microconcentrator.
Sandwich ELISA for MCP-1 Serial dilution titration analyses were performed to determine optimal concentations of both capture and detection antibodies using a fixed amount of MCP-1. Based on these results, microtitre plates were coated with 3 p,g/ml capture antibody in coating buffer (50 mM H3B03, 150 mM NcCl, pH 9.6) by incubating overnight at 4°C. All subsequent incubations were carried out at 37°C. Non-specific binding sites were blocked with 300 ~1 1 mglml BSA in coating buffer for 30 min. After washing the plates twice with TBS-Tween buffer, 100 ~1 of either serially diluted standard MCP-1 or various dilutions of test sample were added. The profile of the standard curve was not affected by using either TBS-Tween or RPM1 1640 supplemented with up to 10% FCS as dilution buffer. Following incubation for 90 min, the plates were washed twice with wash buffer and 100 ~1 4 kg/ml biotinylated anti-MCP-1 antibody in TBSTween containing 2% FCS was added for 90 min. Plates were washed twice and all wells received 100 ~1 0.1 pg/rnl streptavidin-alkaline phosphatase in TBS-Tween-2% FCS for 60 min. After five TBS washes (without Tween), 100 ~1 substrate solution were added and the reaction was stopped with 100 ~10.4 M NaOH after optimal colour development. The absorbance was read at 405 nm on a ELISA reader and MCP-1 concentrations were calculated by interpolation from the standard curve.
ELISA~for IL-8 The concentration of IL-8 was determined using a double-ligand immunoassay.66 Briefly, wells of microtitre plates coated with monoclonal mouse anti-IL-8 antibodies were incubated with test samples and recombinant IL-8 standard. Bound IL-8 was detected with polyclonal goat anti-IL-8 antibodies conjugated to alkaline phosphatase
Vol. 6, No. 2 (March 1994: 162-170)
using the substrate p-nitrophenylphosphate. of the IL-8 ELISA was 50 pg/ml.
The sensitivity
Radioimmunoassay (RIA) for the Quanti$cation of PGE A specific RIA as described previously67 was used for the determination of PGE in cell culture supernatants. The results were expressed as total PGE because the assay was performed with polyclonal anti-PGE1 antibodies crossreacting with PGE2. The RIA consistently detected PGE concentrations > 8 pg/ml.
Statistics The significance of differences between groups was assessed by the paired, two-tailed Student’s t-test; results were considered significantly different if P < 0.05.
Acknowledgement The excellent technical assistance of Marianne Zwicker and Andrea Blaser is gratefully acknowledged. This work was supported by the Swiss National Fund for Biological and Medical Research (grant 3231340.91 to MS) and in part by the Kurt & Senta Hermann Foundation.
REFERENCES 1. Harris ED Jr (1990) Rheumatoid arthritis: pathophysiology and implications for therapy. N Engl J Med 322:1277-1289. 2. Dayer J-M, de Rochemonteix B, Burrus B, Demczuk S, Dinarello CA (1986) Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells. J Clin Invest 77:645-648. 3. Dayer J-M, Beutler B, Cerami A (1985) CacheWtumor necrosis factor stimulates collagenase and prostaglandin E, production by human synovial cells and dermal fibroblasts. J Exp Med 162:2163-2168. 4. Alvaro-Gracia JM, Zvaifler NJ, Firestein GS (1990) Cytokines in chronic inflammatory arthritis. V. Mutual antagonism between interferon-gamma and tumor necrosis factor-alpha on HLA-DR expression, proliferation, collagenase production and granulocyte macrophage colony-stimulating factor production by rheumatoid arthritis synoviocytes. J Clin Invest 86:1790-1798. 5. Leizer T, Clarris B, Ash P, van Damme J, Saklatvala J, Hamilton J (1987) Interleukin-lp and interleukin-la stimulate the plasminogen activator activity and prostaglandin E, levels in human synovial cells. Arthritis Rheum 30:.562-566. 6. Guerne P-A, Zuraw BL, Vaughan JH, Carson DA, Lotz M (1989) Synovium as a source of interleukin 6 in vitro. Contribution to local and systemic manifestations of arthritis. J Clin Invest 83:585-592. 7. Leizer T, Cebon J, Layton JE, Hamilton JA (1990) Cytokine regulation of colony-stimulating factor production in cultured human synovial fibroblasts: I. Induction of GM-CSF and G-CSF production by interleukin-1 and tumor necrosis factor. Blood 76:1989-1996.
Effects of anti-rheumatic drugs on MCP-l/IL-8 8. Alvaro-Gracia JM, Zvaifler NJ, Brown CB, Kaushansky K, Firestein GS (1991) Cytokines in chronic inflammatory arthritis. VI. Analysis of the synovial cells involved in granulocyte-macrophage colony-stimulating factor production and gene expression in rheumatoid arthritis and its regulation by IL-1 and tumor necrosis factor-alpha. J Immunol 146:3365-3371. 9. Butler DM, Piccoli DS, Hart PH, Hamilton JA (1988) Stimulation of human synovial fibroblast DNA synthesis by recombinant human cytokines. J Rheumatol 15:1463-1470. 10. Hirota K, Akahoshi T, Endo H, Kondo H, Kashiwazaki S (1992) Production of interleukin 8 by cultured synovial cells in response to interleukin 1 and tumor necrosis factor. Rheumatol Int 12:13-16. 11. Koch A, Kunkel SL, Burrows JC, Evanoff HL, Haines GK, Pope RM, Strieter RM (1991) Synovial tissue macrophage as a source of the chemotactic cytokine IL-8. J Immunol 147:21872195. 12. Golds EE, Mason P, Nyirkos P (1989) Inflammatory cytokines induce synthesis and secretion of gro protein and a neutrophil chemotactic factor but not B2-microglobulin in human synovial cells and fibroblasts. Biochem J 259:585-588. 13. Watson ML, Westwick J, Fincham NK, Camp RD (1988) Elevation of PMN cytosolic free calcium and locomotion stimulated by novel peptides from IL-l-treated human synovial cell cultures. Biophys Biochem Res Commun 155:1154-1160. 14. Villiger PM, Terkeltaub R, Lotz M (1992) Production of monocyte chemoattractant protein-l by inflamed synovial tissue and cultured synoviocytes. J Immunol 149:722-727. 15. Koch, AE, Kunkel SL, Harlow LA, Johnson B, Evanoff HL, Haines GK, Burdick MD, Pope RM, Strieter RM (1992) Enhanced production of monocyte chemoattractant protein-l in rheumatoid arthritis. J Clin Invest 90:772-779. 16. Oppenheim JJ, Zachariae COC, Mukaida N, Matsushima K (1991) Properties of the novel pro-inflammatory supergene ‘intercrine’ cytokine family. Annu Rev Immunol9:617-648. 17. Matsushima K, Oppenheim JJ (1989) Interleukin 8 and MCAF: novel inflammatory cytokines inducible by IL 1 and TNF. Cytokine 1%13. 18. Baggiolini M. Walz A, Kunkel SL (1989) Neutrophilactivating peptide-l/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 84:1045-1049. 19. Zachariae COC, Anderson AO, Thompson HL, Appella E, Mantovani A, Oppenheim JJ, Matsushima K (1990) Properties of monocyte chemotactic and activating factor (MCAF) purified from a human fibrosarcoma cell line. J Exp Med 171:2117-2182. 20. Rollins BJ, Walz A, Baggiolini M (1991) Recombinant human MCP-l/JE induces chemotaxis, calcium flux and the respiratory burst in human monocytes. Blood 78:1112-1116. 21. Bischoff SC, Krieger M, Brunner T. Dahinden CA (1992) Monocyte chemotactic protein 1 is a potent activator of human basophils. J Exp Med 175:1271-1275. 22. Brennan FM, Zachariae COC, Chantry D, Larsen CG, Turner M, Maini RN, Matsushima K, Feldmann M (1990) Detection of interleukin 8 biological activity in synovial fluids from patients with rheumatoid arthritis and production of interleukin 8 mRNA by isolated synovial cells. Eur J Immunol20:2141-2144. 23. Peichl P. Ceska M, Effenberger F, Haberhauer G, Broell H, Lindley IJD (1991) Presence of NAP-l/IL-8 in synovial fluids indicates a possible pathogenic role in rheumatoid arthritis. Stand J Immunol34:333-339. 24. Vane JR (1971) Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature 231:232-235. 25. Sipe JD, Barle LM, Loose LD (1992) Modification of proinflammatory cytokine production by the anti-rheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response. J Immunol 148:480-484. 26. Lozanski G, Ballou SP, Kushner I (1992) Effect of flurbi-
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profen on cytokine production by human monocytes and U-937 and THP-1 cell lines. J Rheumatol19:921-926. 27. Okamoto M, Sasano M, Goto M, Nishioka K, Aotsuka S-I, Nakamura K, Yokohari R (1991) Suppressive effect of antirheumatic drugs on interleukin-1B release from human peripheral blood monocytes. Int J Immunopharmacol 13:39-43. 28. Otterness IG, Bliven ML, Downs JT, Natoli EJ, Hanson DC (1991) Inhibition of interleukin 1 synthesis by tenidap: a new drug for arthritis. Cytokine 3:277-283. 29. Chang D-M. Baptiste P, Schur PH (1990) The effect of anti-rheumatic drugs on interleukin 1 (IL-l) activity and IL-l and IL-l inhibitor production by human monocytes. J Rheumatol 17:1148-1157. 30. Flower RJ, Blackwell GI (1979) Anti-inflammatory steroids induce biosynthesis of a phospholipase A2 inhibitor which prevents prostaglandin generation. Nature 278:456-459. 31. Beato M (1989) Gene regulation by steroid hormones. Cell 56:335-344. 32. Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science 240:889-895. 33. Cronstein BN, Eberle MA, Gruber HE, Levin RI (1991) Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells. Proc Nat1 Sci USA 88:24412445. 34. Hashimoto K, Whitehurst CE, Matsubara T, Hirohata K, Lipsky PE (1992) Immunomodulatory effects of therapeutic gold compounds. Gold sodium thiomalate inhibits the activity of T cell protein kinase C. J Clin Invest 89:1839-1848. 35. Nesher G, Moore TL (1990) The in vitro effects of methotrexate on peripheral blood mononuclear cells. Modulation by methyl donors and spermidine. Arthritis Rheum 33:954-959. 36. Seitz M, Dewald B, Gerber N, Baggiolini M (1991) Enhanced production of neutrophil-activating peptide-l/interleukin-8 in rheumatoid arthritis. J Clin Invest 87:463-469. 37. Seitz M, Dewald B, Ceska M, Gerber N, Baggiolini M (1992) Interleukin-8 in inflammatory rheumatic diseases: synovial fluid levels, relation to rheumatoid factors, production by mononuclear cells, and effects of gold sodium thiomalate and methotrexate. Rheumatol Int 12:159-164. 38. Hopkins SJ, Humphreys M, Hayson MIV (1988) Cytokines in synovial fluid. I. The presence of biologically active and immunoreactive IL-l. Clin Exp Immunol72:422-427. 39. Yocum DE, Esparza L, Dubry S, Benjamin JB, Volz R, Scuderi P (1989) Characteristics of tumor necrosis factor production in rheumatoid arthritis. Cell Immunol 122:131-145. 40. Saxne T, Palladino MA Jr, Heinegard D, Talal N, Wollheim FA (1988) Detection of tumor necrosis factor (Y but not tumor necrosis factor B in rheumatoid arthritis synovial fluid and serum. Arthritis Rheum 31:1041-1045. 41. Peveri P, Walz A, Dewald B, Baggiolini M (1988) A novel neutrophil-activating factor produced by human mononuclear phagocytes. J Exp Med 167:1547-1559. 42. Hamilton JA, Piccoli DS, Cebon J, Layton JE, Rathanaswani P, McCall SR, Leizer T (1992) Cytokine regulation of colony-stimulating factor (CSF) production in cultured human synovial fibroblasts. II. Similarities and differences in the control of interleukin-1 induction of granulocyte-macrophage CSF and granulocyte-CSF production. Blood 79:1413-1419. 43. Brooks PM, Day RO (1991) Non-steroidal antiinflammatory drugs-differences and similarities. N Engl J Med 324:1716-1725. 44. Kremer JM, Galivan J, Streckfuss A, Kamen B (1986) Methotrexate metabolism analysis in blood and liver of rheumatoid arthritis patients. Association with hepatic folate deficiency and formation of polyglutamates. Arthritis Rheum 29:832-835. 45. Gottlieb NL, Smith PM, Smith EM (1972) Tissue gold concentration in a rheumatoid arthritic receiving chrysotherapy. Arthritis Rheum 15:16-22.
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46. Jessop JD, Johns RGS (1973) Serum gold determinations m patients with rheumatoid arthritis receiving sodium aurothiomalate. Ann Rheum Dis 32:228-232. 47. Leonard EJ, Yoshimura T (1990) Human monocyte chemoattractant protein-l (MCP-1). Immunol Today 11:97-101. 48. Kawahara RS, Deng Z-W, Deuel RF (1991) Glucocorticoids inhibit the transcriptional induction of JE, a platelet-derived growth factor-inducible gene. J Biol Chem 266:13261-13266. 49. Standiford TJ, Kunkel SL, Rolfe MW, Evanoff HL, Allen RM, Strieter RM (1992) Regulation of human alveolar macrophage- and blood monocyte-derived interleukin-8 by prostaglandin E2 and dexamethasone. Am J Respir Cell Mol Biol 6:75-81. 50. Mukaida N, Zachariae CCO, Gusella GL, Matsushima K (1991) Dexamethasone inhibits the induction of monocyte chemotactic-activating factor production by IL-l or tumor necrosis factor. J Immunol 146:1212-1215. 51. Poon M, Megyesi J, Green RS, Zhang H, Rollins BJ, Safirstein R, Taubman MB (1991) In vivi and in vitro inhibition of JE gene expression by glucocorticoids. J Biol Chem 266:2237522379. 52. Deleuran B, Kirstensen M, Paludan K, Zachariae C, Larsen CG, Zachariae E, Thestrup-Pederson K (1992) The effect of second-line anti-rheumatic drugs on interleukin 8 mRNA synthesis and protein secretion in human endothelial cells. Cytokine 4:403-409. 53. Mukaida N, Gussella GL, Kasahara T, Ko Y, Zachariae COC. Kawai T. Matsushima K (1992) Molecular analvsis of the inhibition of interleukin-8 production by dexamethasone in a human fibrosarcoma cell line. Immunol75:674-679. 54. Furst DE, Kremer JM (1988) Methotrexate in rheumatoid arthritis. Arthritis Rheum 31:305-314. 55. Segal R, Mazes E, Yaron M, Tartakovsky B (1989) The effects of methotrexate on the production and activity of interleukin-1. Arthritis Rheum 32:370-377. 56. Segal R, Yaron M, Tartakovsky B (1990) Methotrexate: mechanism of action in rheumatoid arthritis. Sem Arthritis Rheum 20:190-199. 57. Lipsky PE, Ziff M (1977) Inhibition of antigen- and mitogen-induced human lymphocyte proliferation by gold compounds. J Clin Invest 59:455-466. \
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58. Littman BH, Carlson PL, Loose LD, Sanders KM (1990) Effects of gold sodium thiomalate and tenidap sodium (CP-66,2482) on a model of macrophage differentiation using HL-60 cells. Arthritis Rheum 33:29-36. 59. Littman BH, Schwartz P (1982) Gold inhibition of the production of the second complement component by lymphokinestimulated human monocytes. Arthritis Rheum 25:288-296. 60. Sanders KM, Carlson PL, Littman BH (1987) Effects of gold sodium thiomalate on interferon stimulation of C2 synthesis and HLA-DR expression by human monocytes. Arthritis Rheum 30:1032-1039. 61. Matsubara T, Ziff M (1987) Inhibition of human endothelial cell proliferation by gold compounds. J Clin Invest 79:14401446. 62. Matsubara T, Saegusa Y, Hirohata K (1988) Low-dose gold compounds inhibit fibroblast proliferation and do not affect interleukin-1 secretion by macrophages. Arthritis Rheum 31:12721280. 63. Wolf RE, Hall VC (1988) Inhibition of in vitro proliferative response of cultured T lymphocytes to interleukin-2 by gold sodium thiomalate. Arthritis Rheum 31:176-181. 64. Arnett FC, Edworthy Sm, Bloch DA, McShane DJ, Fries JF, Cooper NA, Healey LA, Kaplan SR, Liang MH, Kuthra HS, Medsger TA, Mitchell DM, Neustadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL, Hunder GG (1988) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324. 65. Koski JR, Poplak DG, Blaese RMA (1976) Non-specific Esterase Stain for the Identification of Monocytes and Macrophages. Academic Press, New York, p. 359. 66. Bazzoni F, Cassatella MA, Rossi F, Ceska M, Dewald B, Baggiolini M (1991) Phagocytosing neutrophils produce and release high amounts of neutrophil-activating peptide Uinterleukin 8. J Exp Med 173~771-774. 67. Seitz M, Deimann W, Gram N, Hunstein W, Gemsa D (1982) Characterization of blood mononuclear cells of rheumatoid arthritis patients. I. Depressed lymphocyte proliferation and enhanced prostanoid release from monocytes. Clinical Immunology and Immunopathology 25:405-416.
Beatrice Dewald,2 Marco Baggiolini,2
Michael Seitz’
Activated synoviocytes are major effector cells in the pathogenesis of rheumatoid arthritis (RA) because of their capacity to secrete a variety of inflammatory mediators. Among these mediators, the chemotactic proteins monocyte chemoattractant protein 1 (MCP-1) and interleukin 8 (IL-S) are likely to contribute to the recruitment of inflammatory cells into the arthritic joint. We examined the effects of anti-rheumatic drugs on the MCP-1 and IL-8 production by cultured RA synoviocytes exposed to pro-inflammatory agonists. Both chemotactic cytokines were quantified by specific enzyme-linked immunosorbent assays (ELISA), and found to accumulate in the culture supernatants. Although the time course of formation was similar, the yield of IL-8 was three to IO-fold higher than that of MCP-1. Non-steroidal anti-inflammatory drugs inhibited the synthesis of prostaglandins, but did not influence the production and release of both chemotactic cytokines. Of three disease-modifying drugs tested, dexamethasone and gold sodium thiomalate (GST) inhibited the production of IL-8 and MCP-1, while methotrexate (MTX) was inactive. Dexamethasone reduced the production of MCP-1 and IL-8 by 2045% and 60-SO%, respectively, whilst GST inhibited MCP-1 and IL-8 synthesis in suboptimally, but not in optimally stimulated synoviocytes. Taken together, these results show that the production of MCP-1 and IL-8 is similarly affected by anti-rheumatic drugs and that dexamethasone is the most potent inhibitor suggesting that part of the anti-rheumatic action of glucocorticoids is due to prevention of accumulation of chemotactic cytokines acting on neutrophils and monoctyes.
Rheumatoid arthritis (RA) is a chronic articular inflammatory disorder characterized by synovial lining cell hyperplasia and infiltration of inflammatory cells.’ Synoviocytes of the hyperplastic lining layer exhibit an activated phenotype, and are believed to contribute to the invasive and erosive properties of the inflamed tissue. Cultured synoviocytes exposed to cytokines, such as interleukin 1 (IL-l) and/or tumour necrosis factor (Y (TNF-a), secrete a variety of mediators of inflammation and tissue destruction
From the ‘Department of Rheumatology, University of Bern, Inselspital, CH-3010 Bern, Switzerland, and ‘Theodor-Kocher Institute, University of Bern, CH-3000 Bern, Switzerland. Correspondence to: Dr Pius Loetscher, Department of Rheumatology, University of Bern, Inselspital, CH-3010 Bern, Switzerland. Received 21 April 1993; revised and accepted for publication 12 August 1993 0 1994 Academic Press Limited 1043-4666/94/020162+09 $0800/O KEY WORDS: Anti-rheumatic thritiskynoviocytes 162
drug/IL-S/MCP-l/Rheumatoid
ar-
including collagenase,2-4 plasminogen activator,5 prostaglandin E2 (PGE2),2,4’5 interleukin 6 (IL-6),6 granulocyte-macrophage colony-stimulating factor (GM-CSF)“’ and granulocyte-CSF (G-CSF) ,7 and show increased proliferation.4,9 In addition, several recent studies have demonstrated that synoviocytes also release interleukin 8 (IL-8)10-13 and monocyte chemoattractant protein-l (MCP-1)14’15 in response to cytokine stimulation. IL-8 and MCP-1 belong to a family of structurally related, inducible proteins with four conserved cysteine residues. l&l7 The family is divided into two subfamilies according to the position of the first two cysteines which can be adjacent as for MCP-1 (CC) or separated by one amino acid as for IL-S (CXC). MCP1 and IL-S have different target cell selectivity. IL-8 acts preferentially on neutrophils,i’ whereas MCP-1 activates monocytes and basophils. 19-21 These biological properties together with the finding that MCP1 and IL-S are present at elevated concentrations in RA joint fluids15,22,23 suggest that these chemoattractants contribute to the recruitment of monoCYTOKINE,
Vol. 6, No. 2 (March), 1994: pp 162-170
Effects of anti-rheumatic
nuclear and polymorphonuclear phagocytes into the inflamed synovium. Modulation of MCP-1 and IL-8 production is, therefore, likely to influence disease activity. Non-steriodal anti-inflammatory drugs (NSAID), glucocorticoids, methotrexate (MTX) and gold compounds are widely used to treat rheumatoid arthritis. The mechanisms of action of these drugs is only partly understood. While NSAID act primarily by inhibiting anti-inflammatory cyclo-oxygenase,24 additional effects may be attributed to interference with proinflammatory cytokine production.25-29 Glucocorticoids inhibit production of prostanoids by blocking phospholipase A2,30 but also regulate the expression of a variety of genes.31’32 The mechanism of the disease modifying effects of MTX and gold compounds is still unclear .33-35 We have recently demonstrated that the production of IL-8 by peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis is reduced by dexamethasone, MTX and gold sodium thiomalate (GST), and slightly increased by indomethacin.36,37 We have now studied the effects of a number of representative anti-rheumatic drugs on the production of IL-8 and MCP-1 by stimulated synoviocytes. Furthermore, we examined whether these two closely related chemotactic proteins are differentially modulated by the action of these agents.
I
I
Figure
1.
I
I
I
1
0
Standard
of Antigenic
Production of MCP-1 and IL-8 by RA Synoviocytes In agreement with former reports,lO~ll~ l5 we found that synoviocytes in culture release MCP-1 and IL-8 upon stimulation with LPS, IL-l&, IL-ll3 and
I,
1
curve
of the sandwich
I
4 ELBA
I
5 for MCP-1.
Each point represents the mean of 22 separate determinations carried out in duplicate and vertical bars indicate standard deviation. Since colour development was allowed to proceed for various periods of time, the optical density values at 405 nm were normalized to the absorbance obtained with 5 rig/ml MCP-1. 100% correspond to values ranging from 1.79 to 2.48. The lower limit of detection was 37 p&/ml (P = 0.02). Identical standard curves were obtained with chemically synthesized and recombinant MCP-1.
1.
Specificity 0
MCP-1
A standard curve representing a compilation of 22 separate assays is shown in Fig. 1. The lower limit of detection of MCP-1 was about 40 pg/ml. To assess selectivity, several structurally related cytokines as well as IL-1B and TNF-or were tested for crossreactivity. As shown in Table 1, none of the control cytokines was detected by the sandwich ELISA. Similar negative results were obtained in a direct ELISA (data not shown). The reproducibility of the assay is indicated by the coefficients of variation of absorbance at different MCP-1 concentrations, which ranged from 4.6 to 6.6% and 9.7 to 16.4% in intra- and interassay analyses, respectively.
I
2 3 MCP-1 (rig/ml)
of sandwich
Cytokines
Detection
/ 163
100
TABLE
RESULTS
drugs on MCP-l/IL-8
0.1
Absorbance 1.0
ELISA (405 nm)d 5.0
for MCP-1. 10.0
50.0
0.142 MCP-1 RANTES MIP-la MIP-ll3 IL-8 PF4 CTAP-III NAP-2 GROW GROP GROr IPlO IL-1p TNF-a a The cytokine
0.169 0.156 0.141
0.385 0.140 0.142
0.152 0.155 0.144 0.150 0.148 0.148 0.146 0.149
0.155 0.149 0.148 0.150 0.150 0.147 0.146 0.150
0.139 0.145 0.140 0.147 0.141 0.146 0.154 0.141 0.142 0.142 0.146
0.148
0.144
2.089 0.148
0.137 concentration
(~/ml)
is indicated.
TNF-a. Both chemotactic cytokines steadily accumulated in the culture supernatants for up to 10 days as shown in Fig. 2 for synovial cells stimulated with ILll3 and TNF-a. Similar results were also obtained with IL-la and LPS (data not shown). Maximal levels of MCP-1 and IL-8 were released after stimulation with
164 I Loetscher
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et al.
T
Vol. 6, No. 2 (March 1994: 162-170)
tially modulate the production of colony-stimulating factors in synovial fibroblasts.42 Therefore, we examined whether common NSAID with different plasma half-lives (tiaprofenic acid < indomethacin < naproxen < piroxicam43) also affect the release of IL8 and MCP-1. As shown in Fig. 3, all NSAIfi strongly decreased PGE production without appreciably affecting the release of MCP-1 and IL-K Although the yield of both cytokines tended to rise with the concentration of NSAID added, the increase was not statistically significant. MCP-1 and IL-S release by stimulated synovial cells were also unaffected by the addition of exogenous PGE,, even at high concentrations (data not shown). These results indicate that cycle-oxygenase products are not involved in the regulation of these two chemotactic cytokines in synoviocytes, and that NSAID do not modulate MCP-1 and IL-8 production.
Effect of Dexamethasone
0 Figure
2.
Time
2 course
4 of MCP-1
6 Days and IL-8
8 release
by
10 synoviocytes.
RA synoviocytes were cultured in RPMI-1% FCS in the absence (A) or presence of 10 rig/ml IL-lp (0) or TNF-ol (m). MCP-1 and IL-S were determined in the culture supernatants by ELBA. Mean values k SEM for cells from three RA patients.
l-10 rig/ml IL-1 or TNF-(r. These concentrations are closely comparable to those observed in synovial fluids from patients with RA.38-40 IL-l was always more potent than TNF-a (Fig. 2) or LPS. Synoviocytes produced about three to 10 times more IL-S and MCP-1 irrespective of the inducing agent. As formerly reported for IL-8,41 MCP-1 was found to be remarkably resistant to proteolytic degradation. Over 95% of antigenic MCP-1 added to synovial cell cultures could be recovered after 10 days of incubation (data not shown).
Effect of Non-steroidal
Anti-inflammatory
Drugs
NSAID are still the mainstay of therapy for rheumatoid arthritis. It has been shown that they differen-
Glucocorticoids are potent anti-inflammatory agents and are often used intra-articularly to treat synovitis. Hence, the effect of the synthetic glucocorticoid dexamethasone on the expression of IL-S and MCP-1 by synoviocytes in response to suboptimal (0.01 rig/ml) and optimal stimulatory concentrations (1 rig/ml) of IL-la and -p, TNF-a and LPS was investigated. As shown in Fig. 4, addition of dexamethasone (0.01 and 1 PM) significantly decreased the production of both cytokines. The inhibitory effect was similar for all stimuli and concentrations used. The production of IL-8, however, was inhibited more markedly than that of MCP-1 (P < 0.05; Fig. 4). These results together with the data obtained with NSAID suggest that the inhibitory action of dexamethasone is not mediated by a change in prostanoid release, but rather by effects on gene expression.
Effect of Methotrexate Thiomalate
and Gold Sodium
Treatment of synoviocytes with increasing concentrations of methotrexate followed by stimulation with suboptimal (0.01 rig/ml) or optimal concentrations (1 rig/ml) of IL-lp did not affect the production of either MCP-1 or IL-S (Fig. SA). MTX concentrations corresponding to serum levels observed therapeutically (0.01 and 1 pg/ml)44 as well as an excessive concentration (50 kg/ml) were tested. In contrast, GST (l-50 pg/ml) significantly reduced the production of both MCP-1 and IL-8 in RA synoviocytes stimulated with a suboptimal concentration of IL-lp (Fig. 5B). Inhibition was observed at concen-
Effects
A
of anti-rheumatic
drugs
on MCP-l/IL-8
I 165
100, Stimulus:
0.01
ngiml
II
Stimulus:
1.0 ngiml
Stimulus:
0.01 ngiml
Stimulus:
1.0 rig/ml
150 g $ z8 c
100
2 B
50
I””
0
hi z 100 2
e Figure 3. Effect of NSAID on MCP-1, l@ stimulated RA synoviocytes.
0 IL-8
and PGE release
by IL-
The cells were cultured for 1 h in RPMI-10% FCS with or without drug before addition of IL-lp (10 rig/ml). After 72 h MCP-1, IL-8 and PGE were assayed in the culture supernatants. Percent release with respect to control (no NSAID) is shown: 0,O.l PM; q , 1 PM; q , 10 PM; 0, 100 pM. Control values (100%) were 9 to 47 rig/ml for MCP-1, 73 to 217 &ml for IL-S and 6 to 26 @ml for PGE. Mean values + SEM for cells from four RA patients. t P < 0.05 or 0.02, * P < 0.005 or 0.001.
None IL-la Figure 4. Effect of dexamethasone 8 release by RA synoviocytes.
IL-1S (dex)
TNF-(Y LPS on (A) MCP-1 and (B) IL-
The cells were cultured for 2 h in RPMI-1% FCS with or without dexamethasone (0.01 or 1 KM) before addition of indicated stimulus (0.01 or 1 q/ml). After 72 h MCP-1 and IL-S concentrations were determined in the culture supernatants. Percent release with respect to control (no dex) is shown: q , 0.01 PM; 8,l.O ~.LM. Mean values k SEM for cells from four RA patients. P < 0.05 for MCP-1 release and P < 0.005 for IL-8 release.
166 I Loetscher
CYTOKINE,
et al.
MCP-1
0.01
1
0.01
1
IL-1B (ngiml) Figure 5. late (GST) synoviocytes.
Effect of methotrexate on MCP-1 and IL-8
(MTX) release
and gold sodium thiomaby IL-@ stimulated RA
The cells were cultured for 2 h in RPMI-1% FCS with or without MTX or GST and then stimulated with 0.01 rig/ml or 1 rig/ml IL-1B. 0.01 pg/ml; ma.1 pglml; g, SO kg/ml MTX. (B) q . 10 kg/ml; B. 50 kg/ml GST. After 72 h MCP-1 and IL-8 were assayed in the culture supernatants. Percent release with respect to control (no drug) is shown. Control values (100%) were as follows: 0.01 nglml IL-l@, 9-18 ngiml MCP-1 and 26-143 ngiml IL-S; 1 ng/ ml IL-ll3,20-45 rig/ml MCP-1 and 194-273 rig/ml IL-8. Mean values + SEM for cells from four RA patients. Significant differences are indicated (t. P < 0.02; $, P < 0.01; #, P < 0.001).
trations that are attained in the serum and synovial tissue of GST-treated RA patients.45%46 The effect on IL-8 production was more pronounced than that on MCP-1. No inhibitory effect, however, was detected when the cells were exposed to a IL-lp concentration that resulted in maximal release of the cytokines (Fig. 5B).
DISCUSSION This study was designed to explore the potential role of commonly used anti-rheumatic drugs in reduc-
Vol. 6, No. 2 (March 1994: 162-170)
ing the production of MCP-1 and IL-S by RA synovial cells. MCP-1 and IL-8 are major mediators of inflammation and joint destruction released, by activated synoviocytes in the proliferating synovium. Their main biological function, leukocyte chemotaxis and activation,16’18’47 suggest that they play an important role in the pathogenesis of rheumatoid arthritis. Having confirmed that synoviocytes produce substantial amounts of MCP-1 and IL-8 in response to pro-inflammatory agonists, we studied the effects of anti-rheumatic drugs. NSAID inhibited prostaglandin synthesis in a concentration dependent fashion, but they had no significant effect on MCP-1 and IL-S production. Similarly, it has been reported that NSAID did not affect MCP-1 mRNA levels in mouse fibroblasts.48 In peripheral blood mononuclear cells, however, IL-8 production was increased in the presence of indomethacin.36 Recent studies have reported that various NSAID inhibit the production of other proinflammatory cytokines including IL-l, TNF-a and IL-6 by activated mononuclear phagocytes,25-29 and differentially regulate the levels of colony-stimulating factors in synovial fibroblasts.42 Together, these findings suggest that the actions of NSAID are cell-type and cytokine specific. Some of the effects of these drugs may be mediated by a decrease of prostanoid levels, while others appear to be unrelated to inhibition of cycle-oxygenase. In fact, in the present study added PGE2 did not influence MCP-1 and IL-8 production by synoviocytes and only minimal effects on IL-8 expression by lipopolysaccharide-stimulated alveolar macrophages were observed by others.49 On the other hand, PGE2 inhibited the expression of IL-8 at the mRNA and protein level in peripheral blood monocytes.49 In contrast to NSAID, the synthetic glucocorticoid dexamethasone suppressed the production of MCP-1 and IL-8 by RA synoviocytes exposed to proinflammatory cytokines or bacterial endotoxin. The ineffectiveness of NSAID suggests that the inhibition by dexamethasone is mediated at the level of gene expression rather than indirectly through prostanoids. Glucocorticoids have recently been shown to inhibit the induction of MCP-1 by IL-l or TNF-u in human fibrosarcoma cells5’ and by serum and dibutyryl CAMP in human synoviocytes.‘4 Similarly, glucocorticoids blocked the induction of JE, the murine homolog of MCP-1, by serum or platelet-derived growth factor in cultured vascular smooth muscle cells’i and 3T3 fibroblasts.48 Several mechanisms for the regulation of MCP-1 gene expression by glucocorticoid have been suggested including mRNA destabilizatiot?l and/or inhibition of transcription.48,50 Glucocorticoids also inhibit the expression of IL-8,52,53 an effect that was attributed to inhibition of gene transcription in a human fibrosarcoma cell line.53
Effects of anti-rheumaticdrugs on MCP-l/IL-S / 167
MTX is a highly effective drug for the treatment of RA.54 Yet, we did not observe any significant regulatory activity of this drug on the production of MCP1 and IL-8 by RA synoviocytes. Likewise, no effect on IL-8 release from endothelial cells has been reported.52 In PBMC from RA patients, we previously observed an inhibition of IL-8 production by MTX3’ in contrast
to Deleuran
et al. who found
no sup-
pression of IL-8 secretion.52 It was recently reported that MTX inhibits the activity of IL-1 released from stimulated human PBMC without affecting IL-1 production.29,55 Several effects may contribute to the action of MTX, but the mechanism of its anti-
rheumatic activity remains a mystery.56 Several effects of gold compounds, such as GST, on monocytes have been reported. They include inhibition of accessory cell function,57 differentiation into effector cells,58 decreased production of complement
components59 and decreased expression of HLA-DR in response to IFN. 6o In addition, GST was shown to inhibit IL-l activity29 and IL-8 release.37 Monocytes, however, are not the only target of GST. As shown by the present work, GST, at concentrations that are attained in the serum and synovium of RA patients,
significantly decreased MCP-1 and IL-8 release from synoviocytes under moderate stimulatory conditions (0.01 rig/ml IL-lp). GST has previously been shown to inhibit the proliferation of endothelial cells61 fibroblasts and lymphocytes,62,63
and to modulate
IL-8
mRNA expression and protein secretion.52 These studies indicate that GST affects a variety of cell types and different cellular activities. The biochemical basis for these various effects, however, has still to be elucidated.34 In conclusion, among several anti-rheumatic drugs examined, dexamethasone exhibited the most potent inhibitory effect on the production of MCP-1 and IL-8 by synoviocytes derived from patients with rheumatoid arthritis. A decrease in the synthesis of chemotactic proteins in the inflamed synovium may be one of the mechanisms by which glucocorticoids exert their anti-inflammatory
rheumatoid
MATERIALS
actions in diseases such as
arthritis.
AND METHODS
Reagents Penicillin-streptomycin-fungizone, RPMI-1640, Escoli lipopolisaccharide (LPS), and trypsin-EDTA were purchased from Gibco BRL (Gaithersburg, MD), protein G Sepharose was from Pharmacia-LKB (Uppsala, Sweden), alkaline phosphatase-conjugated goat anti-rabbit antibodies and alkaline phosphatase substrate kit were from Bio-Rad Laboratories AG (Richmond, CA). Fetal calf
cherichia
serum (FCS) was obtained from Amimed (Muttenz, Switzerland). Dexamethasone, prostaglandin EIz,indomethacin, and collagenase type II from Clostridium histolyticum were obtained from Sigma (St Louis, MO). Naproxen was supplied by Syntex Laboratories, piroxicam by Pfizer (Groton, CT), gold sodium thiomalate by Byk Gulden (Byk AG, Kreuzlingen, Switzerland) and methotrexate by Cyanamid AG (Zurich, Switzerland). Streptavidin-alkaline phosphatase, NHS-LC-biotin was purchased from Pierce (Rockford, IL) and MaxiSorb microtitre plates were from Nunc (Roskilde, Denmark). IL-& NAP-2, CTAP-III, PBP, Grocu, B, y, PF4, IPlO and MCP-1 were chemically synthesized and generously provided by Dr I. Clark-Lewis, University of British Columbia, Vancouver, Canada. Recombinant MIPlo, MIP-ll3 and RANTES were kindly provided by Dr C.A. Dahinden, Department of Clinical Immunology, University of Bern, Bern, Switzerland, with the permission of T. Schall, Genentech Inc., So. San Francisco, USA. IL-la and IL-ll3 were generously gifts from Dr K. Vosbeck, CibaGeigy AG, Basel, Switzerland, and TNF-or was kindly supplied by Hoffmann-La Roche, Basel, Switzerland.
Synovial Cell Cultures Synovial tissue was obtained from RA patients undergoing total shoulder, knee or hip joint replacement, who were selected according to the American Rheumatoid Association criteria. 64 Fresh synovial tissue was minced and digested in RPM1 1640 medium containing 1% fetal calf serum (FCS), penicillin-streptomycin-fungizone and 1 mg/ ml collagenase at 37°C until dissolution of the tissue fragments. After filtration through a nylon mesh, the cells were washed with phosphate-buffered saline (PBS) and cultured in RPM1 1640 supplemented with 10% FCS and antibiotics in a humidified COz atmosphere. The following day, nonadherent cells were removed and the medium was replaced. Upon reaching confluence, the cells were harvested with trypsin/EDTA, and subcultures were established. The content of macrophage-like cells determined by staining for non-specific esterase65ranged from 3 to 13%. Synoviocytes from passage 3 to 6 were used for all experiments. They were transferred to 24-well plates (lo5 cells per well) in RPM1 1640 containing 10% FCS. After 24 h, the cells were washed twice with PBS and incubated with 1 ml RPM1 1640 supplemented with 1% FCS (if not otherwise indicated). Drugs were added to the cultures 1 or 2 h before stimulation with pro-inflammatory agonists. NSAID, PGEz and dexamethasone were dissolved in ethanol. The final ethanol concentration did not exceed 0.1% which had no effect on synoviocyte viability as assessedby trypan blue exclusion. Culture supernatants collected at the indicated times were stored -70°C until assayedfor IL-8, MCP-1 and PGE.
Anti-MCP-1
Antibodies
Rabbit polyclonal antibodies were obtained by intradermal injection of synthetic MCP-1 (220 kg) emulsified with complete Freund’s adjuvant followed by three booster injections at weekly intervals. Anti-MCP-1 antibodies in the serum were determined by a direct ELISA. Microtitre plate
168 I Loetscher
CYTOKINE,
et al.
wells (Nunc-Immuno Plate Maxisorb) were coated with 100 ng MCP-1 in 100 ~1 PBS, pH 7.2, overnight at 4°C. After blocking non-specific binding sites with 300 ~1 1 mg/ml BSA in PBS for 30 min at 37”C, the plates were rinsed three times with wash buffer (TBS-Tween: 0.05 M tris-HCl, 0.15 M NaCl, pH 7.5, containing 0.05% Tween 20 and 0.02% sodium azide), and incubated with 100 pi/well of antiserum serially diluted in TBS-Tween. Following incubation at 37°C for 90 min, the plate was washed twice with wash buffer and 100 ~1 of a 1:2000 dilution of alkaline phosphatase conjugated to goat anti-rabbit IgG in wash buffer were added. The plate was incubated for 30 min at 37°C and, after five washes with TBS (without Tween 20), 100 ~1 of substrate solution (1 mg/ml p-nitrophenylphosphate in diethanolamine buffer) was placed in each well. After optimal colour development at 37”C, the reaction was stopped by the addition of 100 ~1 0.4 M NaOH and the absorbance was measured at 40.5 nm in an ELISA reader. The IgG fraction of high titre antiserum (1:lOOOOO) was isolated by chromatography on protein G Sepharose. For use as detecting antibody, a portion of the purified IgG fraction was biotinylated with NHS-LC-Biotin according to the manufacturer’s instruction. Unreacted NHS-LC-biotin was removed by centrifugation using a Centricon 30 microconcentrator.
Sandwich ELISA for MCP-1 Serial dilution titration analyses were performed to determine optimal concentations of both capture and detection antibodies using a fixed amount of MCP-1. Based on these results, microtitre plates were coated with 3 p,g/ml capture antibody in coating buffer (50 mM H3B03, 150 mM NcCl, pH 9.6) by incubating overnight at 4°C. All subsequent incubations were carried out at 37°C. Non-specific binding sites were blocked with 300 ~1 1 mglml BSA in coating buffer for 30 min. After washing the plates twice with TBS-Tween buffer, 100 ~1 of either serially diluted standard MCP-1 or various dilutions of test sample were added. The profile of the standard curve was not affected by using either TBS-Tween or RPM1 1640 supplemented with up to 10% FCS as dilution buffer. Following incubation for 90 min, the plates were washed twice with wash buffer and 100 ~1 4 kg/ml biotinylated anti-MCP-1 antibody in TBSTween containing 2% FCS was added for 90 min. Plates were washed twice and all wells received 100 ~1 0.1 pg/rnl streptavidin-alkaline phosphatase in TBS-Tween-2% FCS for 60 min. After five TBS washes (without Tween), 100 ~1 substrate solution were added and the reaction was stopped with 100 ~10.4 M NaOH after optimal colour development. The absorbance was read at 405 nm on a ELISA reader and MCP-1 concentrations were calculated by interpolation from the standard curve.
ELISA~for IL-8 The concentration of IL-8 was determined using a double-ligand immunoassay.66 Briefly, wells of microtitre plates coated with monoclonal mouse anti-IL-8 antibodies were incubated with test samples and recombinant IL-8 standard. Bound IL-8 was detected with polyclonal goat anti-IL-8 antibodies conjugated to alkaline phosphatase
Vol. 6, No. 2 (March 1994: 162-170)
using the substrate p-nitrophenylphosphate. of the IL-8 ELISA was 50 pg/ml.
The sensitivity
Radioimmunoassay (RIA) for the Quanti$cation of PGE A specific RIA as described previously67 was used for the determination of PGE in cell culture supernatants. The results were expressed as total PGE because the assay was performed with polyclonal anti-PGE1 antibodies crossreacting with PGE2. The RIA consistently detected PGE concentrations > 8 pg/ml.
Statistics The significance of differences between groups was assessed by the paired, two-tailed Student’s t-test; results were considered significantly different if P < 0.05.
Acknowledgement The excellent technical assistance of Marianne Zwicker and Andrea Blaser is gratefully acknowledged. This work was supported by the Swiss National Fund for Biological and Medical Research (grant 3231340.91 to MS) and in part by the Kurt & Senta Hermann Foundation.
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